The present application relates to the detection of single-molecules and the measurement and analysis of single-molecule kinetics and thermodynamics.
Studies at the single molecule level have revealed intramolecular dynamics and conformational changes in many biomolecular systems. The intramolecular chain diffusion of nucleic acids, including the hairpin configuration, has been studied by optical techniques such as fluorescence correlation spectroscopy (FCS). In certain studies, labels are attached to the DNA hairpin and the opening and closing rates of a small number of molecules can be monitored at sub-microsecond time-scales. One of the potential disadvantages of FCS, however, can be that observation time is limited to the diffusion time of molecules through the observation volume. Single-molecule fluorescence resonance energy transfer (smFRET) has also been used to study conformational changes in biomolecules but provides only tens of millisecond time-scales for kinetic studies. Label-free technologies for biomolecular detection include nanowires, microcavities, mechanical cantilevers, optical waveguides and optical tweezers, but none have combined high enough sensitivity for label-free detection with the high temporal resolution necessary to monitor the kinetics of biomolecular processes to microsecond time-scales.
Certain single-molecule-based sequencing-by-synthesis (SBS) systems have become commercially used because they can function without amplification, simplifying sample preparation. Because of their use of fluorescence, certain system designs involves complex trade-offs in the design of the dyes and dye chemistries, laser excitation systems, optics and filtering, and detector characteristics. These challenges can stem from the use of photons as an intermediary for detection, when ultimately an electrical signal is required by the detection electronics.